US20230165776A1 - Protease formulation for treatment of microbial infections - Google Patents

Protease formulation for treatment of microbial infections Download PDF

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US20230165776A1
US20230165776A1 US17/998,059 US202117998059A US2023165776A1 US 20230165776 A1 US20230165776 A1 US 20230165776A1 US 202117998059 A US202117998059 A US 202117998059A US 2023165776 A1 US2023165776 A1 US 2023165776A1
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protease
hyaluronic acid
glycerol
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Mats Clarsund
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CLARTECH AB
Zymiq Technology AB
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/4826—Trypsin (3.4.21.4) Chymotrypsin (3.4.21.1)
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
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    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
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    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
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    • A61Q17/02—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings containing insect repellants
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Definitions

  • the present invention relates to a stable protease formulation for the treatment of microbial infections.
  • Microbial adhesion on the host cell membrane is the most common mode of establishing an infection in the host.
  • Microbial surface protein recognizes cell surface protein that initiates microbial infection via adhesion, internalization, colonization, and secretion of toxic molecules enabling bacterial cell development and growth within the host tissue (Meena et al. 2020).
  • a good strategy to treat and prevent microbial infections could be to reduce or prevent the first step in infection, microbial adhesion.
  • microbes Surfaces of mammalian bodies that are exposed to the external environment are colonized by microbes, the microbiota.
  • Some of the endogenous microbes can, under certain circumstances, cause disease as well as some of the exogenous pathogens that live in the external environment, or on other animals, can overcome our antimicrobial defences and do us harm if we come into contact with them.
  • a good strategy to convert a pathogenic dysbiotic microbiota to a healthy symbiotic microbiota could be to target biofilm structures and the adhesion mechanism to reduce overgrowth of pathogens and to increase microbial diversity.
  • Several studies have identified differences in the microbes present in diseased skin versus those present in healthy skin (Rosenthal et al. 2011) and indicates that an imbalance of microorganisms, termed dysbiosis, exists in numerous pathologies.
  • Antibiotics are also commonly found in veterinary treatment of animals, where many of the drugs used are the same ones used in the treatment of bacterial infections in humans, and antibiotic resistance are associated with treatment failures and subsequent poor animal health. Companion animals are also able to acquire and exchange multidrug resistant pathogens with humans, and may serve as a reservoir of antimicrobial resistance for in-contact people (Buckland et al, 2016).
  • Biofilms are communities of microorganisms protected by a self-synthesised layer of complex polysaccharides, proteins, lipids, and extracellular DNA.
  • Antibiotics are commonly employed systemically to inhibit the growth of pathogenic bacteria and will during treatment act on a range of bacterial species in our commensal microbiota, which is of importance for our health. This can lead to a reorganisation of resident communities, including decreased bacterial diversity and outgrowth by previously minor contributors in gut and skin microbial populations.
  • the human skin and mucosa for example, is a complex barrier organ made of a symbiotic relationship between microbial communities in constant dialogue with the host's innate and adaptive immune system, a well-controlled but delicate equilibrium, mandatory for a healthy skin. Antibiotic-induced alterations of healthy microbiota can persist for several months or years post-treatment (Shaw et al, 2019).
  • Enzymes, and particularly proteases are very suitable to reduce or inhibit microbial adhesion, such as bacterial adhesion, and to dissolve the biofilm without killing microbials.
  • Proteases have been used in medicine for several decades and is an established and well-tolerated class of therapeutic agents (Craik et al, 2011).
  • the inventors of the present invention have developed a novel formulation.
  • Said formulation is a stable protease formulation capable of preventing and reducing biofilm formation as well as to increase microbial diversity in/on a subject or biological surface. Furthermore, said formulation does not induce microbial resistance and can therefore be used instead of antibiotics. Thus, the formulation described herein carry high potential for clinical use and provides a solution to the challenge of reducing the use of antibiotics.
  • the disclosure relates to said composition for use in medicine.
  • the disclosure relates to a composition
  • a composition comprising
  • the disclosure relates to a composition
  • a composition comprising
  • the disclosure relates to a method for prevention and/or reduction of biofilm, wherein the method comprises administration of a composition according to any one of the preceding claims.
  • the disclosure relates to a method for increasing microbial diversity, wherein the method comprises administration of the said composition.
  • the disclosure relates to a method of manufacturing the composition described herein above, comprising mixing the components at 25° C., in the following order:
  • FIG. 1 Formulation optimisation increases the stability of trypsin in solution. Plots displaying the effect of various formulations on the remaining activity of trypsin after storage at (40 ⁇ 2° C./100% RH).
  • Formulation composition (N16) contains glycerol 60 w/w %, CaCl 2 ) 0.1 w/w %, Collagen 0.3 w/w % and 0.04 w/w % trypsin at pH 8.5.
  • Formulation composition (N15) contains glycerol 50 w/w %, CaCl 2 ) 0.1 w/w %, Collagen 0 w/w % and 0.04 w/w % trypsin at pH 8.5.
  • the (Buffer) solution contained 0.04 w/w % trypsin at pH 7.5. Non-linear regression was used to estimate half-life.
  • CFU colony-forming units
  • FIG. 4 There was a clear correlation between abundance of clinically relevant pathogens and microbial diversity.
  • FIG. 6 The alpha diversity was primarily increased in the enzyme group. Plots displaying the alpha diversity, i.e. the average species diversity in the treated area, on (A) day 0 and (B) day 28. At day 28, the mean alpha diversity had increased from 197 to 404 (median; from 53 to 479) in the enzyme treatment group and for the chlorhexidine group a small non-significant increase from 359 to 392 (median; from 345 to 388) was observed. There was no apparent difference in the control group (mean; from 300 to 312, median; from 300 to 192). Data is plotted as means with error bars (standard error).
  • FIG. 8 There was a prolonged positive effect displayed in the dogs treated with Formulation A. The gingivitis status testing was continued also after the study ended, and the score continued to stay low even more than 60 days post-initiation. This effect appeared to persist for more than 60 days, suggesting that the non-healthy microbiota had been replaced by a healthier microbiota.
  • protease as used herein means an enzyme that cleaves a polypeptide of any length, short or long, at a peptide bond, for example by hydrolysing the peptide bond.
  • the substrate of a protease is thus a polypeptide.
  • the substrate can be a naturally occurring polypeptide or a synthetic polypeptide.
  • biofilm denotes an extracellular matrix in which microorganisms are dispersed and/or form colonies.
  • the biofilm typically is made of polysaccharides and other macromolecules.
  • the phrases “preventing a biofilm” “reducing a biofilm”, or “decreasing a biofilm”, and like phrases means the prevention of biofilm growth, reduction in the rate of biofilm growth, partial eradication of existing biofilm, and/or complete eradication of existing biofilm.
  • stability refers to in vivo stability and storage stability (e.g., storage stability at room temperature).
  • microbial diversity refers to a measure of how many different types of microbial species there are in a within a particular environment, such as the mouth or on the skin of a mammal, while at the same time taking into account the overall representation of the species relative to other species.
  • w/w means a “weight/weight”.
  • % w/w is synonymous to “wt %” or “weight-percent”.
  • 10 g of a composition comprising 50% w/w of A comprises 5 g A.
  • phrases such as “a composition comprising X to Y % of A” are taken to mean a composition comprising A in the range of X to Y %, both thresholds included. That is, the composition does not comprise less than X % of A, and the composition does not comprise more than Y % of A, provided X is smaller than Y.
  • the present disclosure relates to a stable protease formulation for treatment of microbial infections.
  • Proteases are very suitable in treatments against microbial infections, biofilm, and are non-toxic and well-tolerated as a therapeutic agent. It is also an advantage with a formulation that do not induce resistance and that can be used instead of antibiotics.
  • the divalent cation of said composition is calcium.
  • the composition comprises 0.01 to 0.2% of a calcium salt.
  • the composition comprises 0.01 to 0.2% calcium chloride.
  • the composition further comprises 0.1 to 0.4% w/w buffer, e.g. a pH buffer.
  • the buffer may be a mixture of two or more buffer systems.
  • the buffer of said composition is selected from the group consisting of Tris (2-Amino-2-(hydroxymethyl)propane-1,3-diol) and MOPS (3-Morpholinopropane-1-sulfonic acid).
  • Tris (2-Amino-2-(hydroxymethyl)propane-1,3-diol) and MOPS (3-Morpholinopropane-1-sulfonic acid) A person skilled in the art will appreciate the use of other pharmaceutically acceptable buffers.
  • the composition comprises essentially no monosaccharide, such as 0.0% w/w monosaccharide.
  • the monosaccharide of said composition is mannose.
  • the composition comprises 0.0 to 0.2% w/w mannose, such as 0.01 to 0.2% w/w mannose.
  • the composition further comprises collagen.
  • the collagen may be any collagen extracted and purified from tissues of animals and fishes and shellfishes. The extraction and decomposition method or the modification degree of the resulting collagen is not specifically limited.
  • the collagen may be modified, such as chemically modified.
  • the collagen is gelatin.
  • the composition comprises 0.0 to 0.3% w/w collagen, such as 0.01 to 0.3% w/w collagen.
  • the formulation may advantageously comprise collagen.
  • Collagen may improve stability of dermal compositions, and may have further advantageous properties for dermal use.
  • the composition comprises collagen, such as 0.01 to 0.3% collagen.
  • the composition comprises essentially no collagen, such as 0.0% w/w collagen.
  • the protease of said composition is trypsin. Trypsin is a serine protease which predominantly cleaves peptide chains at the carboxyl side of the amino acids lysine and arginine.
  • the composition comprises 0.01 to 0.2% w/w protease, such as 0.01 to 0.2% w/w trypsin.
  • the composition comprises 0.02 to 0.09% w/w protease, such as 0.02 to 0.09 w/w trypsin.
  • the composition comprises essentially 0.04% w/w protease, such as 0.04% w/w protease.
  • the composition comprises one or more of a diol, a triol, or a polyol, such as glycerol, propylene glycol, or a sugar alcohol.
  • the composition comprises a diol.
  • the composition of the disclosure comprises 50 to 80% w/w of the diol, such as 50 to 75% w/w, such as 50 to 70% w/w, such as 50 to 62% w/w.
  • the composition comprises 50 to 80% w/w glycerol, such as 50 to 75% w/w, such as 50 to 70% w/w, such as 50 to 62% w/w.
  • the composition comprises essentially 60% diol, such as glycerol.
  • the role of the diol, triol, and/or polyol is in part to reduce the water activity in the composition. Reducing the water activity in turn deactivates the protease. Deactivation of the protease improves its stability in the composition.
  • the composition comprises 0.0 to 0.04% w/w glycosaminoglycan, such as hyaluronic acid. In another specific embodiment, the composition comprises 0.002 to 0.04% w/w glycosaminoglycan, such as hyaluronic acid. In another specific embodiment, the composition comprises essentially 0.02% w/w glycosaminoglycan, such as hyaluronic acid. In one embodiment of the present disclosure, the composition comprises 0.0 to 0.1% w/w hyaluronic acid, such as 0.002 to 0.1% w/w.
  • composition comprises or consists essentially of:
  • said composition comprises or consists essentially of:
  • said composition comprises or consists essentially of:
  • the combination of components of said composition does not exceed 100%.
  • the pH of said composition is between 6.0 to 7.5, such as 6.2 to 7.4.
  • pH of solutions can be assessed in a number of ways. For example, pH can be assessed using a pH electrode or a pH indicator.
  • the composition further comprises water.
  • at least part of the water of the composition originates from the buffer.
  • the presence of free water can activate and/or destabilize the protease.
  • Water in the composition may be deactivated by other components, such as for instance glycerol and other alcohols describes herein.
  • the composition comprises less than 50% w/w water, such as 20 to 50% w/w water.
  • it is important that the water activity in the composition is low.
  • water from said wound, skin, or oral cavity mixes with the composition, increasing the water activity. This in turn activates the protease.
  • the composition is the form of a spray, a gel, a cream, lotion, an ointment, a foam, or a dental chew.
  • the disclosure relates to said composition for use in medicine. Specifically, one embodiment provides for the disclosed composition for use in medicine.
  • the disclosure provides a composition comprising
  • the disclosure provides a composition comprising
  • the microbial infection is selected from the group consisting of bacterial infections, fungal infections, yeast infections and viral infections.
  • the dermatological condition is a skin infection.
  • the dermatological condition is an inflammatory skin disease.
  • the inflammatory skin disease is selected from the group consisting of atopic dermatitis, psoriasis, rosacea and acne.
  • the oral condition is an oral infection. In one embodiment, the oral condition is a dental infection. In another embodiment, the mammal is a dog, a cat, a horse or a human.
  • the protease of said composition is trypsin.
  • the divalent cation of said composition is calcium.
  • composition further comprises 0.1 to 0.4% w/w buffer.
  • the buffer of said composition buffer is selected from the group consisting of Tris and MOPS.
  • the composition further comprises a monosaccharide.
  • the monosaccharide of said composition is mannose.
  • the composition further comprises collagen.
  • the pH of said composition is between 6.0 to 7.5, such as 6.2 to 7.4.
  • the composition further comprises water.
  • the composition is the form of a spray, a gel, a cream, lotion, an ointment, a foam, or a dental chew.
  • the composition is for topical use.
  • the present disclosure relates to a method for prevention and/or reduction of biofilm, wherein the method comprises administration of a composition according to any one of the preceding claims.
  • the biofilm is a biofilm in or on a mammal.
  • the biofilm is a biofilm of a biological surface.
  • the present disclosure relates to a method for increasing microbial diversity, wherein the method comprises administration of the said composition.
  • compositions that stimulate the skin microbiome and microbial diversity could be a great use in the treatment of such conditions, and for cosmetic purposes.
  • the stable formulation of the present disclosure were found to have similar positive effect on reduction of clinically relevant pathogens as chlorhexidine.
  • chlorhexidine can cause allergic reactions (especially so in eczema patients), that cross-resistance to antibiotics have been reported and that it has limited effect against fungi and mycobacteria.
  • chlorhexidine for dental use can cause side effects such as staining of teeth, increased calculus formation and negative effects on the oral (healthy) microbiome. Therefore, a person skilled in the art will appreciate that the use of the formulation of the present disclosure would be beneficial over chlorhexidine.
  • the disclosure relates to a method of manufacturing the said composition comprising mixing the components at 25° C., in the following order:
  • One embodiment of the present disclosure provides for a method of treating a condition selected from the group consisting of microbial infections, dermatological conditions and oral conditions, in and/or on a mammal, said method comprising administering a composition comprising:
  • the formulation mixes were put into sealed polypropylene bottles. Products from each formulation composition were placed on stability at a stressed condition (40 ⁇ 2° C./100% RH) for up to 411 days. Samples were pulled at predetermined time points and analysed directly by measuring the enzyme activity. The trypsin activity was measured at 30° C. using Z-Gly-Pro-Arg-pNA as substrate in a spectrophotometer at 405 nm. The decrease in absorbance per minute was used as activity parameter. The half-life or remaining activity relative to time point zero was determined and used to describe the stability. Non-linear regression was used to estimate half-life.
  • the half-life for trypsin increased from 2 days in buffer to more than 400 days in the most stable formulations.
  • the half-life could not be accurately determined in the most stable formulations since more than 88% of the enzyme activity still remained after 411 days.
  • the remaining activity after storage at 40° C. for 411 days varied from 20 to 88% for the tested combinations ( FIG. 1 ).
  • Formulation A was also very stable with a remaining activity of >90% after storage at 40° C. for 119 days. Its shelf life at room temperature 25-30° C. was estimated to be more than 3 years.
  • the aim of this study was to evaluate the test formulation, Formulation A, whose stability was tested in Example 1, against preformed biofilms as well as against biofilm formation using the MBECTM Assay by following the ASTM E2799-12 standard.
  • the ASTM E2799-12 is a standard test method for testing disinfectant efficacy against Pseudomonas aeruginosa, Staphylococcus pseudintermedius and Malassezia pachydermatis biofilm using the MBEC assay.
  • a mature biofilm was established on pegs under batch conditions with very low shear produced by gentle rotation of the device on an orbital shaker.
  • the pegs containing the biofilm were rinsed to remove planktonic cells and the peg lid was placed in a receiver plate.
  • the wells in the receiver plate were filled according to an experimental design that contained the appropriate sterility, growth, and neutralizer controls as well as the disinfectants.
  • the disinfectants were a formulation described in Table 2 and a negative control, which was a carrier solution with no active agent (Table 3).
  • the peg lid was placed in a receiver plate containing neutralizer, and the entire device was placed in a sonicator to remove the biofilm and disaggregate the clumps. Samples from each well were then diluted, plated and the viable cells counted. The log reduction in viable cells was calculated by subtracting the mean log density for the treated biofilm from the mean log density for the untreated controls. The results were measured in colony-forming units (CFU)/peg.
  • CFU colony-forming units
  • Formulation A successfully decreased the biofilm formed by tested microbes.
  • the aim of this clinical study was to compare two different solutions on the effect of the natural microbiota in skin folds of dogs.
  • Denatonium benzoate is a bittering agent, which was added to Formulation A to prevent dogs from licking on the wound or treated area.
  • the dog Underwent general clinical examination. Cytology samples were taken from bilateral nasomaxillary folds. Swabs were collected from the dogs' nasomaxillary folds and sent for next generation DNA sequencing (MiDOG, CA, USA) according to manufacturer's instructions. On day 0, the pet owners were also informed of how to apply the test solution (enzyme treatment solution or chlorhexidine) on each nose fold twice daily (morning and evening) until the return visit to the clinic, except for the day of the visit. Two bottles of test product were provided to the owners, and they were instructed to store the test product at room temperature.
  • test solution enzyme treatment solution or chlorhexidine
  • the topical treatment increased the diversity of bacterial and fungal compositions over time, shown as an increase in microbial diversity score ( FIG. 3 ).
  • the enzyme treatment group (Formulation A) the increase was 38%, for the chlorhexidine group 11% and for the control group it was ⁇ 5%.
  • the aim of this study was to evaluate the treatment effect of Formulation A in order to improve the dental status of dogs.
  • the OraStrip® Dental Diagnostic test (CET OraStrip, Virbac, Ft. Worth, Tex.) is a test to detect periodontal disease and to assess the risk of moderate to severe periodontal disease that can be performed at the clinic.
  • the diagnostic test determines level of thiol, an organosulfur compound produced by the bacteria associated with periodontal disease and as a measure of level of gingivitis where 0 is no thiol detected (no gingivitis) and 1-5 indicates active and increasing gingivitis.
  • the dog underwent a relevant clinical examination at the clinic.
  • the clinical examination included a dental status evaluation using the OraStrip® Dental Diagnostic test (on saliva) according to the manufacturer's instructions.
  • a periodontal scoring was also performed in order to assess the degree of gingivitis and periodontitis (Table 4). Photos were taken of the dogs' teeth and gum.
  • the pet owners were informed of how the Formulation A should be applied twice daily (morning and evening) until the return visit, and supplied with an instructions document. Each owner was supplied with one bottle of Formulation A, and informed that it should be stored at room temperature.
  • Periodontal scoring of the dogs PD Score/ Stage Classification Clinical manifestation 0 Clinically No gingival inflammation or periodontitis normal clinically evident 1 Slight Gingivitis only without attachment loss. gingivitis The height and architecture of the alveolar margin are normal 2 Early ⁇ 25% of attachment loss at most. There is periodontitis a stage 1 furcation involvement in multi- rooted teeth and early radiologic signs of periodontitis.
  • the loss of periodontal attachment is less than 25%* 3 Moderate 25-50% of attachment loss*, or a stage 2 periodontitis furcation involvement in multi-rooted teeth 4 Advanced ⁇ 50% of attachment loss*, or a stage 3 periodontitis furcation involvement in multi-rooted teeth *As measured either by probing of the clinical attachment level, or radiographic determination of the distance of the alveolar margin from the cemento-enamel junction relative to the length of the root
  • the OraStrip® test shows the level of gingivitis had decreased for the group treated with Formulation A, indicating a decreased risk of a more severe periodontal disease ( FIG. 7 A ).
  • the periodontal score had also decreased for the dogs treated with Formulation A, suggesting an improved dental health among the treated dogs ( FIG. 7 B ).
  • the level of gingivitis was also tested after the treatment period ended, and the score continued to stay low even more than 60 days after treatment stop ( FIG. 8 ).
  • Formulation A appears to increase the dental health of the dogs, with decreased gingivitis and periodontal score. Furthermore, this effect appears to persist for more than 60 days. This suggests that the non-healthy microbiota has been replaced by a healthier microbiota.

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